Wh
y F
IB/S
EM
?W
hy n
ot
cry
o?
Sam
ple
pre
para
tion
Refe
ren
ces
Ackn
ow
led
gem
en
ts
Univ
ers
ity o
f C
op
enhag
en
B.
Lub
elli
, D
.A.M
. d
e W
inte
r, J.A
. Po
st,
R.P
.J.
van
Hees,
M.R
. D
rury
, C
ryo-F
ib-S
EM
an
d M
IP s
tud
y o
f p
oro
sity
and
pore
siz
e d
istr
ibu
tion
of
bento
nit
e a
nd
kaolin
at
diff
ere
nt
mois
ture
conte
nts
, A
pp
lied
Cla
y S
cience
, 2
01
3,
80
-81
, p
p.
35
8-3
65
J.C
. R
iem
ers
ma,
Osm
ium
Tetr
oxid
e F
ixati
on o
f Li
pid
s: N
atu
re o
f th
e R
eact
ion P
rod
uct
s, J H
isto
chem
Cyto
chem
19
63
11
: 4
36
OsO
4 fi
xati
on
Usi
ng
th
e p
revio
usl
y d
esc
rib
ed
pre
para
tion m
eth
od
im
ag
es
were
ta
ken s
how
ing
thre
e d
iffere
nt
ph
ase
s:
Liq
uid
ph
ase
(lip
id)
Bulk
phase
(ch
alk
)
Air
bu
bb
les
Veri
fyin
g t
he o
bserv
ati
on
s:
Air
bu
bb
les
ind
icate
the o
bse
rvati
on o
f a fi
xed
liq
uid
Ele
menta
l co
ntr
ast
due t
o d
iffere
nt
den
sity
of
lipid
and
bu
lk
Info
rmati
on c
an b
e g
ath
ere
d f
rom
2D
im
ag
es a
s w
ell a
s f
rom
3D
im
ag
e
sta
cks a
cquir
ed
by F
IB/S
EM
.
Fu
ture
possib
ilit
ies:
Dir
ect
ob
serv
ati
on o
f fluid
dis
trib
uti
ons
for
diff
ere
nt
satu
rati
ons
as
well
as
the p
oss
ibili
ty o
f ele
menta
l m
ap
pin
g.
Than
ks t
o t
he N
an
oG
eoS
cience
gro
up
at
Cop
enhag
en
Univ
ers
ity f
or
enab
ling
this
work
in t
he f
ram
ew
ork
of
the P
-cub
ed
pro
ject
.
Sp
eci
al th
anks
to Z
hila
Nik
rozi
and
Kla
us
Qvort
rup
fro
m t
he C
ore
Faci
lity f
or
Inte
gra
ted
Mic
rosc
op
y (
CFI
M)
Cop
en
hag
en.
Imag
ing
liq
uid
s in
pore
s -
b
eyon
d X
-ray t
om
og
rap
hy
Lipid
Fix
ati
on in p
oro
us
sam
ple
s
Resu
lts
Ralp
h H
art
i, H
enn
ing
Søre
nse
n,
Kim
Dalb
y,
Susa
n S
tip
pN
ano-S
cience
Cente
r, U
niv
ers
ity o
f C
op
enh
agen,
Univ
ers
itets
park
en 5
, 2
10
0 K
øb
enhavn,
Denm
ark
Focu
sed
Ion
Beam
- S
can
nin
g
Ele
ctron
Mic
roscop
y (
FIB
/SEM
)
FIB
/SEM
sa
mple
pre
para
tion
befo
re
and
aft
er
rem
ovin
g s
lices
usi
ng t
he F
ocuse
d I
on
Beam
-
the
dir
ecti
on
of
the
imagin
g
ele
ctr
on b
eam
is
indic
ate
d b
y t
he a
rrow
FIB
/SEM
is c
om
bin
ing t
he im
agin
g c
apabilit
ies o
f th
e S
cannin
g E
lectr
on M
icro
scope w
ith
the s
am
ple
m
anip
ula
tion o
pport
un
itie
s o
f th
e F
ocused Ion
B
eam
.
Imag
ing
ste
ps:
1)
Cu
ttin
g a
flat
surf
ace
of
the
sam
ple
(FI
B)
2)
Take
imag
e of
flat
surf
ace
(SEM
)
3)
Rem
ove
thin
slic
e (F
IB)
5)
Con
tinue
wit
h s
tep
2
e-b
eam
Oft
en t
om
og
rap
hy is
use
d t
o a
cquir
e 3
D im
ag
es o
f p
oro
us
mate
rials
(se
e c
en
tral im
ag
e).
Dra
wb
ack
s of
this
tech
nolo
gy
how
ever
are
the lim
ited
num
ber
of
imag
ing
mod
es
and
th
e
exp
ensi
ve a
nd
com
ple
x im
ag
e a
cquis
itio
n.
This
work
pre
sents
a n
ew
sam
ple
pre
para
tion
meth
od
for
imag
ing
lip
ids
in p
oro
us
syst
em
s usi
ng
FIB
/SEM
, overc
om
ing
the lim
itati
ons
of
tom
og
rap
hy a
nd
tack
ling
som
e c
halle
ng
es
of
the c
lass
ical S
EM
ap
pro
ach
.
FIB
/SEM
off
ers
:
Fast
im
ag
e a
cqu
isit
ion (
can
be d
one a
t all
FIB
/SE
M s
etu
ps)
Hig
h s
pati
al re
solu
tion
Mu
ltip
le im
ag
ing
mod
es
(BS
E,
SE
, ED
X)
How
ever
FIB
/SEM
is
a d
est
ruct
ive m
eth
od
lim
itin
g t
he d
ata
colle
ctio
n t
o a
one-t
ime p
oss
ibili
ty.
The d
irect
ob
serv
ati
on
of
the p
ore
sp
ace
, m
ad
e p
oss
ible
by
FIB
/SEM
, and
its
conte
nt
elim
inate
s th
e n
eed
for
imag
e
reco
nst
ruct
ion a
nd
off
ers
a d
irect
vie
w o
n t
he a
rea o
f in
tere
st.
Imm
ob
ilizi
ng
liq
uid
s b
y f
reezi
ng
th
em
is
a c
om
mon a
pp
roach
to im
ag
e
liquid
s in
th
e S
can
nin
g E
lectr
on
Mic
roscop
e (
SEM
). H
ow
ever
com
bin
ing
it
wit
h t
he ion m
illin
g p
roce
ss o
f th
e F
IB/S
EM
is,
esp
eci
ally
consi
deri
ng
poro
us
rock
sam
ple
s, a
challe
ng
ing
task
.
The m
ost
pop
ula
r ap
pro
ach
for
cryo-S
EM
im
ag
ing
of
rock
s is
a
fractu
re p
rocess incl
ud
ing
fra
cturi
ng
the s
am
ple
insi
de t
he S
EM
by
the u
se o
f a b
lad
e (
B.
Lub
elli
et
al.,
20
13
).
Fra
ctu
re t
ech
niq
ue:
No 3
D in
form
ati
on
Top
og
rap
hic
contr
ast
due t
o t
he n
ot
suffi
cientl
y fl
at
surf
ace
aft
er
fract
uri
ng
Fixati
ng
flu
ids
wit
hou
t th
e n
eed
of
freezin
g t
hem
(usi
ng
the
tech
niq
ue illu
stra
ted
in t
his
work
) enab
les
the u
se o
f th
e F
ocu
sed
Ion
Beam
pro
vid
ing
the c
ap
ab
iliti
es
of
3D
info
rmati
on a
s w
ell
as flat
surf
ace
s w
ith
out
top
og
rap
hic
con
trast
.
Ori
gin
al im
ag
eB
ulk
colo
ure
dPo
res
colo
ure
dA
ir b
ub
ble
s co
loure
d
Osm
ium
tetr
oxid
e (
OsO
4) fixati
on is
wid
ely
use
d
in L
ife S
cience
s to
im
mob
ilize
lip
ids
by r
eact
ing
wit
h
unsa
tura
ted
bond
s in
fatt
y
aci
ds
to f
orm
dio
ls.
Oxid
ati
on o
f O
sO4
leadin
g t
o t
he
fixati
on o
f u
nsa
tura
ted lip
ids
(aft
er
J.C
. R
iem
ers
ma, 1
96
2)
Osm
ium
tetr
oxid
e
(OsO
4)
Reacti
on
ste
ps:
Op
en
ing
up
dou
ble
bond
s
C
ycl
oad
dit
ion t
o f
orm
osm
ate
est
er
Rap
id h
yd
roly
sis
lead
ing
to v
icin
ol d
iol
Th
e s
am
ple
To t
ake
ad
van
tag
e o
f th
e p
rinci
ple
of
Osm
ium
Tetr
oxid
e F
ixati
on t
he
sam
ple
has
to b
e fl
ush
ed
wit
h a
lip
id c
onta
inin
g a
suffi
cient
num
ber
of
unsa
tura
ted
fatt
y a
cid
s. S
unfl
ow
er
oil
is
a s
uit
ab
le c
hoic
e.
Em
erg
e s
am
ple
in S
unflow
er
Oil
Heat
up
to a
round
80
°C f
or
an h
ou
r
Take
the s
am
ple
out
Put
sam
ple
in O
smiu
m T
etr
oxid
e g
ase
ous
en
vir
onm
ent
(~ 2
d
ays)
Aft
er
follo
win
g t
his
pro
ced
ure
the lip
id insi
de t
he p
ore
s is
fixe
d a
nd
ca
n b
e t
reate
d a
s a s
olid
.
Basi
c kn
ow
led
ge a
bout
the invest
igate
d c
halk
sam
ple
(Aalb
org
outc
rop
) is
gath
ere
d b
y d
ata
analy
sis
on
tom
og
rap
hic
im
ag
es
(see c
entr
al im
ag
e).
Poro
sity
: ~
50.9
%
Sp
ecific
Su
rface
Are
a: ~
0.6
21 m
2/g
Perm
eab
ility
: ~
32.2
4 m
D
The p
erm
eab
ility
is
calc
ula
ted
usi
ng
th
e K
oze
ny-
Carm
an
eq
uati
on,
i.e.
base
d o
n t
he p
oro
sity
and
sufa
ce a
rea.
3D
im
age d
ata
set
wit
h fi
xed liq
uid
insi
de o
f pore
s
Recommended
